Paroxysmal nocturnal hemoglobinuria in a patient post COVID‐19 virus infection: A case report with literature review

Abstract COVID 19 is a serious infection that originated in Wuhan, China and has resulted in worldwide morbidity and mortality. It continues to be a major health concern in 2022, being associated with multiorgan failure. Although the pathophysiology of the disease and its complications are not well understood, it is believed that a cytokine storm, triggered by complement activation may be responsible for the severity and complications of the disease. As of now, there is no definitive treatment available. Hematological changes associated with COVID‐19 include lymphopenia, anemia, thrombocytopenia, disseminated intravascular coagulopathy, and thrombosis. Paroxysmal nocturnal hemoglobinuria (PNH), on the other hand, is an acquired clonal hematopoietic stem cell disorder that occurs due to an acquired PIG‐A mutation affecting the hematopoietic stem cells. Interestingly, PNH exhibits some clinical and laboratory manifestations like those seen in COVID‐19. In this report, we present a rare case of PNH that developed following a COVID‐19 infection.


| INTRODUCTION
Coronaviruses are a large family of zoonotic viruses that cause a range of illnesses from common flu-like symptoms to severe respiratory disease.Several strains of coronaviruses have caused diseases in humans, including COVID-19, severe acute respiratory syndrome (SARS-CoV), and Middle East respiratory syndrome (MERS-CoV). 1,2he coronavirus outbreak, which began in December of 2019, originated in Wuhan, China.The virus is believed to have been transmitted from animals to humans.According to the World Health Organization (WHO), the main modes of transmission are through air-droplets, aerosol, or direct contact between individuals.The incubation period of the virus which refers to the time from exposure to the onset of symptoms, typically ranges from 1 to 2 weeks. 1,2The pathophysiology of COVID-19 and its associated complications is still not clearly understood.Current publications suggest that complement activation plays a significant role in the pathogenesis of COVID-19.The complement system functions to eliminate immune complexes, facilitate opsonization, induce cell lysis, and initiate an inflammatory process.The complement system consists of three main pathways: classical, alternative, and lectin pathways.Each pathway is stimulated through specific mechanisms and has proximal components (C1-C3) and terminal components (C5-C9).These pathways work collaboratively to produce the membrane attack complex (MAC) which includes complements C5-C9. 3,4Complement pathways inhibition rely on two major proteins expressed on the cell membrane of various cells in the body: CD55 and CD59.CD55, previously known as decay accelerating factor (DAF), is a glycosylphosphatidylinositol (GPI) anchored membrane protein widely expressed on different cells.Its main role is to inhibit and accelerate the decay of classical and alternative pathways C3 convertase.CD59, also known as MAC inhibitor, is a GPI-anchored membrane protein that acts on the terminal pathway components.It prevents the assembly of the MAC by inhibiting the insertion of C9 catalyzed by C5B-8 into the lipid bilayer of the pathogen. 3,4aroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired syndrome with a 30% mortality rate within 5 years.PNH is an acquired clonal hematopoietic stem cell disorder caused by an acquired PIG-A mutation that affects the hematopoietic stem cells.This mutation leads to affected blood cell lines that are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins, specifically CD55 and CD59, which are important complement regulatory proteins.PNH shares some similarities to COVID-19 in terms of clinical and laboratory manifestations (Table 1). 5-7

| CASE REPORT
A 28-year-old gentleman, previously in good health, presented 1 month after being diagnosed with COVID 19 infection.He experienced mild COVID-19 symptoms but did not require any treatment as he was asymptomatic.However, a few weeks later, he was presented with severe abdominal pain, necessitating surgical exploration.The CT scan of his abdomen revealed evidence of splenic infarction.Subsequently, he underwent splenectomy, and examination of the spleen specimen confirmed infarction with no signs of malignancy.Due to his anemia and thrombocytopenia, he was referred to our hospital.The laboratory results upon his presentation to our hospital are provided in Table 2.
He had negative Coombs test, indicating non-immunemediated anemia, and thrombocytopenia.Further investigations ruled out antiphospholipid syndrome and microangiopathic hemolytic anemia (MAHA), including TTP/HUS.His repeated COVID-19 test came back negative.A bone marrow biopsy (Figure 1A,B) revealed mild hypocellularity for his age (40%-50%) and trilineage hematopoiesis with a left shift and decreased megakaryocytopoiesis.No lymphoid aggregates, granuloma or atypical infiltrate were observed.
Chromosomal analysis was not conducted as there was no specific indication for it.Peripheral blood flowcytometry was performed to investigate the presence of an underlying PNH clone.The results of the flow cytometry are presented in Table 3 and Figure 2A,B.
These findings are indicative of a diagnosis of PNH.During his hospital stay, the patient experienced complications including intracerebral hemorrhage (ICH), which necessitated surgical evacuation.Unfortunately, eculizumab and other complement inhibitors were not accessible for treatment.Despite receiving intravenous immunoglobulin (IVIG), steroid and supportive blood products the patient's condition deteriorated, and he ultimately succumbed to a massive pulmonary embolism that resulted in cardiopulmonary arrest.

| DISCUSSION
COVID 19 is recognized as a systemic disease with the potential to affect multiple systems, including the hematological system.Hematological changes commonly observed in COVID-19 patients encompass anemia, lymphopenia, thrombocytopenia, and thrombosis.Elevated ferritin levels have been identified as a poor prognostic factor in COVID-19 patients Unfortunately, these changes have often been reported in isolation without being placed within the appropriate context.Complement activation is considered a major contributor to the progression, severity and complications of COVID 19.Deposition of complement components has been documented in various organs, such as the lungs, kidneys, endothelium, and skin, in COVID-19 patients. 5NH is a rare condition that affects approximately 1.13 individuals persons per million in the general population.PNH is an acquired disorder caused by a mutation in the PIG-glycosylphosphatidylinositol (GPI) anchor, affecting proteins like CD55 and CD59.These mutations can be triggered by various factors including viral infections, exposure to chemicals, or radiation, but in many cases the cause is idiopathic.The diagnosis of PNH relies on both clinical presentation and laboratory findings (Table 4).
Clinical presentation of PNH is variable and includes Coombs-negative hemolytic anemia, thrombocytopenia, and thrombosis at unusual site.Patients may also exhibit symptoms and signs such as dyspnea, dysphagia, abdominal pain, erectile dysfunction, renal failure, and pulmonary hypertension.PNH is often associated with other hematologic diseases, particularly bone marrow failure syndromes like aplastic anemia and myelodysplastic syndromes. 8aboratory finding in PNH include Coombs-negative anemia, thrombocytopenia, elevated lactate dehydrogenase (LDH), indirect hyperbilirubinemia, and increased reticulocyte count.However, the current diagnostic criteria for PNH rely on flowcytometry analysis.A diagnosis of PNH requires the demonstration of at least two different GPI protein deficiencies within two different cells lines, such as granulocytes, monocytes, or erythrocytes through flow cytometry. 8,9e laboratory diagnosis of PNH is based on the following three criteria, as recommended by the International PNH Interest Group (IPIG). 91.Evidence of non-immune hemolysis.2. Flow cytometric analysis demonstrating a population of peripheral blood cells deficient in GPI-anchored proteins.Confirmation requires the identification of at least two different GPI protein deficiencies within two different cell lines from granulocytes, monocytes or erythrocytes.3. Bone marrow aspirate, biopsy, and cytogenetics to assess the presence or absence of an associated bone marrow syndrome, such as myelodysplastic syndrome or aplastic anemia.
Various treatments have been employed, including intravenous immunoglobulin and steroids, although their efficacy lacks sufficient evidence. 10The mainstay of PNH treatment involves the use of complement inhibitors, such as eculizumab or ravulizumab, and/or allogenic stem cells transplantation.Despite our patient not receiving these medications due to their high-cost and unavailability at our hospital, we believe they could be the treatment of choice not for PNH but also for covid-19 infection itself.

Monocytes (B)
Several ongoing studies are currently investigating the use of such medications for treating COVID-19 infection.Our case report, along with supporting evidence from publications on complement activation associated with COVID-19 infection, suggests the potential use of complement inhibitors as a treatment for COVID-19 infection.However, the availability, cost, safety, and efficacy of these medications pose limitations.
Currently, there are ongoing clinical trials evaluating different complement inhibitors as a potential treatment for patients with moderate to severe COVID-19 infection.We have summarized the ongoing trials involving complement inhibitors in COVID-19 infection in Table 5. 10

| CONCLUSION
Our case report presents a rare occurrence of PNH following COVID-19 infection, which have been rarely reported in the literature.Considering the limited efficacy and curative potential of currently available treatments, it is worth exploring complement inhibitor medications as an alternative therapy for patients with COVID-19 infection.Conducting large scale trials to assess the effectiveness of these medications is crucial, taking into consideration their cost and availability.Additionally, we recommend studying the incidence of COVID-19-related PNH in patients presenting with COVID-19 infection and cytopenia, as this has significant implications for the treatment of such patients.The data is available on request CONSENT No written consent has been obtained from the patient as there is no patient identifiable data included in this case report/series.

ORCID
U R E 1 Low (A) and high power (B) microscopic image of bone marrow biopsy showing an overall hypocellular marrow for age (10× and 40×).

T A B L E 3 F
Flowcytometry results that showed significant PNH clone.I G U R E 2 (A, B) Selected flowcytometry analysis plots showing the confirmation of loss of GPI anchored proteins from surface of WBCs using (FLAER).